Fig 2: Deletion of PP2A in LepR+ MSCs inhibited proliferation and hypertrophic differentiation of SOC in early postnatal stage.Sections of P15 distal femur from Lepr-cre; Ppp2r1afl/fl mice were subjected to immunostaining. a–d Immunohistochemistry (IHC) and immunofluorescence reveal LepR+ MSCs express Ppp2r1a and unphosphorylated (Y307) PP2AC in SOC. (a) LepR IHC. b Ppp2r1a IHC. c Double immunofluorescence of LepR and Ppp2r1a. d Unphosphorylated (Y307) PP2AC IHC. e IHC reveals successful deletion of Ppp2r1a in LepR+ MSCs in SOC. IHC reveals that deletion of Ppp2r1a in LepR+ MSCs leads to decreased expression of Ki67 (f) and hypertrophic markers, such as Runx2, Osterix, collagen X, MMP13 and alkaline phosphatase (g), and increased expression of Perilipin (h) in SOC at P15. HZ = Hypertrophic zone; POC = Primary ossification center; ColX =collagen X; ALP = alkaline phosphatase. Scale bar, 100 µm.

Fig 3: EREG improved glucose tolerance in the absence of leptin in Lepob mice and exhibited no effect in LepR-deficient Leprdb mice. (A) Body weight of Lepob male mice in groups before and after treatment with Veh (PBS, white bar) or EREG (50 ng/g body weight (BW), black bar) for 26 days. Mice were on regular chow diet. Unpaired t-test, n = 7/group. ns, not significant. (B,C) Fat (B) and lean body (C) composition in same groups of mice at the end of the study was measured by Echo-MRI. Fat and lean mass are shown as % of the total weight (100%). (D,E) Glucose tolerance test (GTT) was performed in fasted Lepob mice treated with PBS (Veh, open circles) or EREG (closed circles) (n = 7 per group). GTT kinetics (D) and area under the curve (AUC) (E) are shown. Statistical significance was examined by ANOVA (D) and Student’s t-test (E). (F) Insulin levels in plasma in both mouse groups were measured by ELISA. Unpaired student’s t-test. (G) Weight before and after treatment of Leprdb male mice with Veh (PBS, white bar) or EREG (50 ng/g body weight (BW), black bar) for 4 weeks (n = 6 per treatment). Mice were on regular chow. Unpaired Student’s t-test, n = 6/group. (H,I) Fat (H) and lean body (I) composition (% of total weight) in the same groups of mice at the end of the study were measured by Echo-MRI. (J,K) GTT kinetics (J) and AUC (K) were obtained from Leprdb mice treated with PBS (Veh, open circles) or EREG (closed circles). ANOVA (J) and Student’s t-test (K). (L) Insulin levels in plasma in both mouse groups were measured by ELISA. Unpaired student’s t-test.

Fig 4: EREG regulated glucose uptake via binding with LepR in Lepob mice. (A) EREG and insulin tolerance test in Lepob mice (n = 5 per group) treated with a single intraperitoneal injection of insulin (0.012 IU/g BW, triangle dashed line) or EREG (80 ng/g BW, closed circles. Asterisks, significant (* p < 0.05) compared to glucose levels before EREG treatment. # Hashtag, significant difference in glucose levels 30 min after treatment with EREG or insulin. Unpaired Student’s t-test. (B) Area under the curve (AUC) quantification of insulin (hatched bar) and EREG (black bar) tolerance tests. Unpaired Student’s t-test, ns. (C) GTT kinetics were measured in Lepob mice (n = 5 per treatment) treated with a single injection of PBS (Veh, open circles) or EREG (closed circles). Student’s t-test. * p < 0.05 from comparison between control and EREG treated mice at each time point. (D) Area under the curve (AUC) quantification of insulin (hatched bar) and EREG (black bar) tolerance tests. Unpaired Student’s t-test. (E,F). Immunoprecipitation of LepR was performed with anti-EREG antibody using homogenates from subcutaneous fat (C) and visceral fat (D). Fat tissue was isolated from non-treated Lepob (Veh) as well as Lepob mice 15 min after injection of EREG (50 ng/mL).

Fig 5: PP2A deletion in LepR+ MSCs increases Runx2 phosphorylation at Ser472 and reduces chondrocyte hypertrophy.MSCs isolated from WT (WT MSCs) and Lepr-cre; Ppp2r1a fl/fl mice (PP2A KO MSCs) were subjected to analysis. a Representative western blot analysis of WT MSCs after osteogenic induction. b Representative western blot analysis of WT MSCs after adipogenic induction. c Western blot analysis of WT and PP2A KO MSCs. d Co-Immunoprecipitation study of cell lysates from WT MSCs with indicated induction for osteogenesis. IP with PP2A-specific or phospho Ser 472 Runx2-specific antibody and precipitates were probed for phospho Ser472 Runx2 and PP2A. e and f IHC analysis of inactive form phospho Ser472 Runx2 expression in hypertrophic chondrocyte of POC (E18.5) and SOC (P15) from Lepr-cre; Ppp2r1a fl/fl mice and relative WT littermate mice. g Quantification of phospho Ser472 Runx2 percentage in hypertrophic chondrocyte of POC (E18.5) and SOC (P15) from Lepr-cre; Ppp2r1a fl/fl mice and relative WT littermate mice. ***p < 0.001 as determined with Student’s t-test. Data are mean ± s.d. h Scheme representing regulation of MSC differentiation by PP2A and its substrates protein. Scale bar, 50 µm.